Type 2 diabetes mellitus, T2D, is a chronic metabolic disorder which is characterized by increased hepatic glucose production, defective pancreatic beta-cell functions, insulin secretion deficiency and insulin resistance, finally leading to the sustained hyperglycemia situations (Green, et al. Current Pharmaceutical Design, 2004, 10). T2D is also the leading cause of kidney failure, blindness and amputation, and is definitely correlated with the high risk of death from cardiovascular causes in the world. Furthermore, the prevalence of T2D is believed to be a growing factor due to the increasing epidemic of obesity, especially in the developing countries. Although many anti-diabetic treatments have been approved by the FDA (the U.S. Food and Drug Administration), new therapeutic strategies are supposed to achieve better glycemic and body weight controls.
La Barre proposed the concept of incretin (intestinal secretion of insulin) in 1929, which initiated a novel therapy for the treatment of diabetes many years later. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are the two most potent incretin hormones in human which have ever been discovered and examined extensively during the last 30 years (Baggio and Drucker, Gastroenterology, 2007, 132, 2131-2157). GLP-1 and GIP are released from intestinal endocrine L and K cells, respectively. GLP-1 is a 30 or 31-amino-acid peptide, which is produced from its precursor pre-proglucaon through post-translational gene processing (Orskov, et al. Endocrinology, 1986, 119, 1467-1475). Meanwhile, GIP is secreted as a 42-amino acid peptide and shares approximately 68% sequence homology with GLP-1 (Gallwitz, et al. Regulatory Peptides, 1996, 63, 17-22).
Both GLP-1 and GIP exhibit their physiological functions through bindings to their specific receptors, GLP-1 receptor (GLP-1R) and GIP receptor (GIPR) respectively, which belong to the G-protein coupled receptor family (Seino, et al. Journal of Diabetes Investigation, 2010, 1, 8-23). GLP-1 and GIP are responsible for stimulating insulin secretion after nutrient ingestion as glucose-dependent profile. This glucose-dependent mode of action offers the much lower possibility of hypoglycemia, when compared to other traditional anti-diabetic therapies such as insulin injection, orally administered sulphonylureas and metformin, etc. Moreover, they also stimulate insulin biosynthesis, improve pancreatic beta-cell proliferation, and inhibit beta-cell apoptosis; hence they potentially preserve beta-cell functions and slow the T2D progression (Green. Best Practice & Research Clinical Endocrinology & Metabolism, 2007, 21, 497-516). All these featured properties are leading to the great interest in exploring GLP-1 and GIP as potential therapeutic agents for T2D and other metabolic disorders.
Although GLP-1 and GIP exhibit anti-diabetic potential, they are rapidly degraded by dipeptidyl peptidease-IV (DPP-IV) into inactive forms [GLP-1 (9-37, or 9-36), and GIP (3-42)] and removed from the circulation in vivo (Deacon, et al. Journal of Clinical Endocrinology & Metabolism, 1995, 80, 952-957). This enzyme specifically digests an alanine, proline or hydroxyproline residue in the penultimate N-terminal position. Therefore, endogenous GLP-1 and GIP have very short half-life times in human (2 min and 5-7 min, respectively) due to DPP-IV degradation (Deacon, et al. Hormone and Metabolic Research, 2004, 36, 761-765). That is the reason why exogenous GLP-1 and GIP can't be applied directly for anti-diabetic treatments.
Many research groups have tried to achieve DPP-IV resistance through structural modifications based on native GLP-1 sequence and GIP sequence. First of all, N-terminal extension by diverse chemical substitutions at His7 and Tyr1 positions of GLP-1 and GIP, respectively (Green, et al. Journal of Endocrinology, 2004, 180, 379-388; O'Harte, et al. Diabetes, 1999, 48, 758-765). Secondly, Ala8 and Glu9 in GLP-1, and Ala2 and Glu3 in GIP respectively, have been widely replaced by other amino acids, or even some unusual amino acids (Green, et al. Journal of Molecular Endocrinology, 2003, 31, 529-540; Biological Chemistry, 2003, 384, 1541-1551; Metabolism, 2004, 53, 252-259; Gault, et al. Journal of Endocrinology, 2003, 176, 133-141; Metabolism, 2003, 52, 679-687; Biochemical & Biophysical Research Communications, 2002, 290, 1420-1426; Diabetologia, 2003, 46, 222-230). Lastly, GLP-1 and GIP have been conjugated with short- or long-chain fatty acids to prolong circulation time and bioavailability (Holz and Chepurny, Current Medicinal Chemistry, 2003, 10, 2471-2483; Irwin, et al. Journal of Medicinal Chemistry, 2005, 48, 1244-1250; Journal of Medicinal Chemistry, 2006, 49, 1047-1054). These structural modifications produced various biological activities, along with much improved stabilization against DPP-IV degradation.
Exendin-4, a peptide comprising of 39 amino acids, was firstly isolated and identified from lizard venom in 1992 (Eng, et al. Journal of Biological Chemistry, 1992, 267, 7402-7405). Exendin-4 shares approximately 53% amino acid homology with GLP-1 sequence, and subsequent experiments demonstrated exendin-4 was a pure and potent GLP-1 receptor agonist in vitro and in vivo. It is no wonder that exendin-4 exhibits quite similar physiological properties to native GLP-1, and also regulates gastric emptying, insulin secretion, food intake, and glucagon secretion. Exendin-4 induces blood glucose reduction in normal rodents and in both mice and rats with experimental diabetes (Raufman, Regulatory Peptides, 1996, 61, 1-18). It is much more potent than native GLP-1 in vivo, mainly because of its improved pharmaco-kinetic properties. It has a glycine substitution at position 2 in the sequence, while alanine is present at the same position in the native GLP-1 sequence. Thus, exendin-4 should be stable for DPP-IV degradation, and has a much longer half-life time than native GLP-1 in vivo. Chemically synthesized exendin-4, exenatide co-developed by Amylin and Eli Lilly, was approved by the FDA in 2005 under the trade name of “Byetta” for the treatment of T2D, due to its potent blood glucose lowering features along with comparatively prolonged circulation of action in vivo. Currently, exenatide has to be administered through subcutaneous injection twice daily. A long-acting form of exendin-4, designated exenatide-LAR has been studied extensively in both animal models and human in once weekly profile (Gedulin, et al. Diabetologia, 2005, 48, 1380-1385; Drucker, et al. Lancet, 2008, 372, 1240-1250).
Encouraged by the great success of exenatide, Novo Nordisk developed liraglutide, a long-acting GLP-1 analog, which shares approximately 97% sequence identity to human GLP-1. Based on native GLP-1 (7-37) sequence, liraglutide has an arginine substitution at position 34, and palmitoyl conjugation to the epsilon-amine of Lys at position 26 with gamma-glutamyl as the spacer. The palmitoyl conjugation is believed to achieve DPP-IV protection and significantly prolong its circulation rate in vivo through binding to serum albumin and peptide self-association (Malm-Erjefält, et al. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 2010, 38, 1944-1953). Under the trade name of “Victoza”, liraglutide was approved by the EMA (the European Medicines Agency) and the FDA in 2009 and in 2010 respectively for the treatment of T2D with the profile of once daily subcutaneous injection. The exploratory studies of liraglutide indication in obese non-diabetic subjects are now in phase III clinical trial (Astrup, et al. Lancet, 2009, 374, 1606-1616). Other GLP-1 and exendin-4 analogs as pure GLP-1 receptor agonists, such as taspoglutide, albiglutide, and lixisenatide etc, are to date under late clinical development stages.
Although GIP is responsible for approximately 60% of the incretin effect in normal subjects (Nauck, et al. Journal of Clinical Endocrinology and Metabolism, 1986, 63, 492-498), the therapeutic applications of GIP analogs for anti-diabetic potential have been largely limited by the diminished or markedly attenuated responsiveness of pancreatic beta-cell towards GIP in some but not all subjects with T2D (Krarup, et al. Metabolism, 1987, 36, 677-682; Jones, et al. Hormone and Metabolic Research, 1989, 21, 23-26). Hence, the reduced insulin secretion induced by GIP was observed after the intravenous administration of the peptide in T2D patients. On the contrary, GLP-1 was shown to stimulate insulin secretion in different stages of T2D subjects effectively (Nauck, et al. Journal of Clinical Investigation, 1993, 91, 301-307).
The reduced insulinotropic effect of GIP was speculated due to chronic desensitization of GIP receptor (Tseng, et al. American Journal of Physiology, 1996, 270, E661-666), or due to the reduced expression of GIP receptor on pancreatic beta-cells in T2D patients (Hoist, et al. Diabetologia, 1997, 40, 984-986). Despite only minor effort has been emphasized on the clinical applications of GIP, it has been demonstrated that GIP contributes to the pathogenesis of T2D to a considerable degree (Meier, et al. Regulatory Peptides, 2002, 107, 1-13). As recently reported, N-terminally modified GIP analogs have been shown to increase insulin response to glucose and lower plasma glucose levels in obese diabetic oblob mice (O'Harte, et al. Journal of Endocrinology, 2000, 165, 639-648).
Furthermore, the reduced release of insulin induced by GIP in T2D is much less evident in pulse administration than in continuous intravenous infusion (Meier, et al. Diabetes, 2004, 53, S220-224). Additionally, the sensitivity of pancreatic beta-cells towards endogenous or exogenous GIP in T2D can be re-established when the hyperglycemia situation is reversed (Piteau, et al. Biochemical and Biophysical Research Communications, 2007, 362, 1007-1012). Thus, the potential application of GIP in the treatment of T2D is now being re-emphasized so far. For example, N-AcGIP alone or in combination with exendin-4 induced significant plasma glucose reduction and improved glucose intolerance in the model of dietary-induced obesity-diabetes (Irwin, et al. Regulatory Peptides, 2009, 153, 70-76).
In consideration of the insulinotropic effects induced by GLP-1 and GIP, it has been demonstrated that the stimulatory action on glucose-dependent insulin release and intracellular cAMP production by GLP-1 and GIP should be additive and synergistic as well (Gallwitz, et al. Journal of Molecular Endocrinology, 1993, 259-268; Siegel, et al. European Journal of Clinical Investigation, 1992, 22, 154-157; Nauck, et al. Journal of Clinical Endocrinology and Metabolism, 1993, 76, 912-917). Many research groups have tried to develop synthetic GLP-1/GIP hybrid peptides to achieve the simultaneous activations of both GLP-1R and GIPR.
Gallwitz reported synthetic GLP-1/GIP chimeric peptides possessing an N-terminal and C-terminal third of one peptide with an exchange comprising the middle portion of the other peptide. Furthermore, hybrid peptides with additional singular mutations in the positions 13 and 15 were prepared as well as chimeres with only an exchange in the C-terminal third. Unfortunately, the binding affinity of these hybrid peptides to GLP-1R was found to be sensitive to GIP-like exchanges in the N-terminal 22 amino acids as well as in positions 13 and 15 (loss of affinity 280-fold to more than 1,000-fold). C-terminal replacement of GLP-1 sequence by GIP reduced the affinity to GLP-1R only 20-fold, and all the hybrid peptides exhibited minimal binding affinity against GIPR (Gallwitz, et al. Endocrinology and Metabolism, 1995, 2, 39-46; Regulatory Peptides, 1996, 63, 17-22).
Hinke also reported a series of five GIP/GLP-1 hybrid peptides to elucidate the binding and activation domains of GIP and GLP-1 in the perspective of the known bioactive domains of GIP. The chimeric peptides synthesized were GLP-1[1-14]/GIP[15-30]NH2 (CH1), GIP[1-14]/GLP-1[15-30]NH2 (CH2), GLP-1[1-11]/GIP[12-30]NH2 (CH3), GIP[1-11]/GLP-1[12-18]/GIP[19-30]NH2 (CH4), and GIP[1-14]/GLP-1[15-18]/GIP[19-30]NH2 (CH5). The subscript amino acid designations for GLP-1 were numbered according to the primary sequence (i.e. GLP-1[7-37]=GLP-1[1-31]). Through GIPR and GLP-1R transfected CHO cell assays, the added GLP-1 sequence in these chimeras did not contribute to the binding affinity or bioactivity of these peptides at the GIPR (Hinke, et al. Life Sciences, 2004, 75, 1857-1870).
The patent, WO 2010011439, described modified glucagon analogs exhibiting potent GIP receptor activation in addition to glucagon and/or GLP-1 activity. Based on glucagon sequence, the hybrid peptides were developed through substitutions of the middle portion (17-19) with GLP-1 sequence, and replacement of the C-terminus with residues from exendin-4 (30-39). It also claimed alpha, alpha-disubstituted amino acids at positions 2 and 20 respectively, as the most important modifications to achieve DPP-IV protection and simultaneous activations of both GLP-1R and GIPR. The dual GLP-1R/GIPR agonists exhibited much more potency than the pure GLP-1 agonists (such as exendin-4 and liraglutide) in the case of blood glucose level reduction and body weight loss in diet-induced obese (DIO) mice. The dual agonists induced body weight loss and blood glucose level reduction even in GLP-1R knock-out model (DiMarchi and Ma, WO 2010011439, 2009, Jun. 16).
Interestingly, it was reported that the simple co-administration of GIP in addition to GLP-1 did not further augment insulin secretion effect, nor did it lead to more blood glucose lowering in patients with T2D (Mentis, et al. Diabetes, 2011, 60, 1270-1276). This may indicate that the simple combination of GIP and GLP-1 could not potentiate the additivity and synergism of the insulinotropic effectiveness induced by individual GIP and GLP-1 clinically. Coincidently, Gault reported that a single preparation of liraglutide-N-AcGIP was more potent at lowering plasma glucose and insulin secretion stimulation when compared to liraglutide and N-AcGIP or a simple peptide combination in normal male NIH Swiss TO mice and in obese diabetic (oblob) mice (Gault, et al. Clinical Science, 2011, 121, 107-117).